Regulation of glucocorticoid receptors in the kidney of immature and mature male rats

1. Specific binding of [3H]dexamethasone to cytosol and the activation of bound hormone-receptor complexes were studied in the kidney of immature (3-week) and mature (26-week) Long-Evans male rats. 2. The concentration of specific binding sites was significantly higher (25%) in the kidney of immature rats as compared with mature, while dissociation constants (Kd) remain unaltered at both ages. 3. Heat activation (25 degrees C for 45 min) significantly enhanced the binding of [3H]dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both ages to the same extent. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice versa) gave similar results to the non-mixed groups. 4. Ca2+ activation (0 degree C for 45 min with 20 mM Ca2+) also enhanced the nuclear and DNA-cellulose binding at both ages but to a greater magnitude in immature rats. 5. Differences in the number of specific binding sites and some of the physicochemical properties of kidney glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.     

Age-dependent activation of glucocorticoid receptors in the liver of male rats

The binding of hepatic [3H] dexamethasone-receptor complexes to DNA-cellulose and purified nuclei was studied in the immature (3-week) and mature (26-week) Long-Evans male rats to determine the age-associated changes, if any, in the physicochemical properties of glucocorticoid-receptors. Our data show that heat activation (for 45 min at 25 degrees C) significantly enhances the binding of [3H] dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both the ages, with a greater magnitude in mature rats. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice-versa) show receptor specificity. Ca2+ activation (20mM Ca2+ for 45 min at 0 degrees C) also enhances the nuclear and DNA-cellulose binding at both the ages but to a similar extent. These findings indicate that some of the physicochemical properties (e.g. heat activation) of glucocorticoid receptor change, while others (e.g. Ca2+ activation) remain unchanged at these phases of the life span. The observed changes may lead to functional alterations in the tissue response as a function of age.     

Regulation of rat liver NADP+-isocitrate dehydrogenase during aging

In an attempt to understand the mechanism of aging in relation to the differences in enzyme regulation, the induction and kinetic properties of NADP⁺ -isocitrate dehydrogenase of the liver of immature (6 weeks), mature (13 weeks), adult (33 weeks) and old (85 weeks) female rats were studied. The specific activity of the cytoplasmic and mitochondrial NADP+ -isocitrate dehydrogenase increased up to the adult age (33 weeks) and decreased in the old rats (85 weeks). Overiectomy decreased and estradiol administration induced activity of both the mitochondrial and eytoplasmic enzyme in the liver ol immature, mature and adult rats but had no significant effect in old rats. However, the activity of mitochondrial NADP⁺ -isocitrate dehydrogenase decreased and eytoplasmic NADP+ -isocitrate dehydrogenase increased following ovariectomy in old rats (85 weeks). Hormone-mediated induction of enzyme activity was actinomycin D sensitive. The Km for isocitrate and NADP, Ki value for oxalomalate, heat stability and electrophoretic mobility of the purified enzyme from the cytosol fraction of the liver of immature and old rats were similar. It can he concluded that the enzyme does not change structurally with age. Part of this work was presented at the 48th Annual General Meeting of the Society of Biological Chemist, India, 1979.     

Effect of post-synthetic modifications of proteins on the binding of estrogen-receptor complex to uterine nuclei of aging rats

The binding of estrogen-receptor (ER) complex to nuclei following post-synthetic modifications of proteins was examined in the uteri of young (18 weeks) and old (96 weeks) rats. Acetylation decreases the binding of ER complex to nuclei but methylation shows no effect on the extent of binding in both ages. On the other hand, phosphorylation enhances the binding of ER complex by two-fold in nuclei from young rats but reduces this to half in nuclei from old rats. The pattern of binding in salt-resistant nuclear fractions is similar to that in total nuclei except in methylation where old rats show about 20% higher binding as compared to the respective control. These findings suggest that post-synthetic modifications of proteins modulate the binding of ER complex to uterine nuclei in an age-specific manner.     

Assessment of “activation” of estrogen receptors in lactating mammary gland using DNA-cellulose binding

To circumvent the need for isolated nuclei in studies on activation of estrogen-receptor complexes in mammary gland of the rat, a DNA-cellulose binding assay was employed using a cell-free system. Incubation at 28°C for 30 min of receptors previously charged with [³H]estradiol markedly enhanced their association with DNA-cellulose. Once activated, estrogen-receptor complexes bound maximally to DNA-cellulose within 20–30 min. The temperature optimum for activation was 28 ± 2°C using cytosol preparations. The temperature-induced activation required the presence of both steroid and cytosolic receptors simultaneously. Density gradient centrifugation revealed that, unlike those of uterus, both activated and charged estrogen-receptor complexes of lactating mammary tissue sedimented as a 4 S species in sucrose gradients containing 0.4 M KCl.     

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.     

Identification of a macromolecular inhibitor of glucocorticoid-receptor complex activation in rat liver cytosol

We have identified an endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE-cellulose. The macromolecular regulator, purified approximately 20-fold as judged by Lowry-reactive material, inhibits activation of glucocorticoid-receptor complexes when assayed by DNA-cellulose binding and by chromatography on DEAE-cellulose minicolumns. In addition the active DEAE-cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE-cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90 degrees C (15 min) and is partially inactivated at 45 degrees C (15-60 min).     

Activated steroid-receptor complex. Comparison of assays using DNA-cellulose or homologous nuclei.

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [3H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the later only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Activated steroid-receptor complex comparison of assays using DNA-cellulose or homologous nuclei

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [³H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the latter only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.     

Inactivation of rat liver glucocorticoid receptor by molybdate. Effects on both non-activated and activated receptor complexes.

When freshly prepared glucocorticoid-receptor complex from rat liver cytosol was incubated at 23 degrees C in the presence of sodium molybdate, its subsequent binding to isolated nuclei, DNA-cellulose and ATP-Sepharose was blocked. In addition, binding to these acceptors by cytosol receptor complex fractionated with (NH4)2SO4 was also blocked by incubation of the complexes with 50 mM-sodium molybdate. However, molybdate had no effect on the binding of activated receptor complexes to ATP-Sepharose. Molybdate was also effective in extracting the nuclear- and DNA-cellulose-bound glucocorticoid-receptor complexes in a dose-dependent manner. Molybdate appears to exert its effects directly on the receptor by interacting with both non-activated and activated receptor forms.     

Changes in glucocorticoid receptors in different regions of brain of immature and mature male rats

Specific cytosolic binding for synthetic glucocorticoid dexamethasone was studied in several brain regions (hypothalamus, hippocampus, caudate nucleus, cerebellum, cerebral cortex) of immature (3-week) and mature (26-week) male rats, intact and adrenalectomized. A significant regional difference was observed in the concentration of in vitro [3H] dexamethasone binding in the brain of adrenalectomized rats at both ages, with the highest levels in the hippocampus. A marked decrease in specific binding was observed in all brain regions of adrenalectomized mature rats as compared to immature. The dexamethasone binding was significantly lower in all brain regions of normal intact animals as compared to adrenalectomized rats in both ages.     

Regulation of NAD- and NADP-linked isocitrate dehydrogenase by hydrocortisone in the brain and liver of male rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) in the brain and liver of rats of various ages were investigated. The activity of NAD-linked isocitrate dehydrogenase of the brain was greater than cytoplasmic or mitochondrial NADP-linked isocitrate dehydrogenase. In contrast, the cytoplasmic NADP-isocitrate dehydrogenase of the liver predominates over both NAD- and mitochondrial NADP-isocitrate dehydrogenases at the three ages studied. The activity of NAD-isocitrate dehydrogenase increased in the brain (139%) and liver (17%) of rats upt o 33 weeks of age and decreased (57 and 39%, respectively) in old rats (85-week-old). The activity of cytoplasmic NADP-isocitrate dehydrogenase was maximum in immature (6-week-old) rat brain and decreased as the age of the rats increased; whereas, in liver, the activity of this enzyme was found to be maximum in adult rats (33-week-old). Brain mitochondrial NADP-isocitrate dehydrogenase activity increased (64%) in adult rats, but in liver it decreased (45 and 33% in 33- and 85-week-old rats, respectively). In both tissues, adrenalectomy and hydrocortisone treatment showed differential age-dependent response. Hydrocortisone-mediated induction of the level of enzymes was inhibited by actinomycin D.     

Postnatal regulation of glucocorticoid receptor in the liver of chicken

The specific binding of [3H]-dexamethasone to glucocoticoid receptor (GR) and activation of hormone-receptor (H-R) complexes from the liver of chicken at day 0, 5, 10, 30, 60 and 90 were investigated to find out GR regulation during postnatal development. Results showed that GR level (fmol/mg protein) reached a peak by day 5 of postnatal age and was significantly higher (+ 42%) than observed at day-0 (day of hatching), as evidenced also by protein blot experiments and Scatchard analysis of binding data. The GR concentration declined gradually up to day 30, and thereafter, no significant change was observed at day 60 and 90 of postnatal ages. The temperature and salt-dependent activation of GR showed no significant differences in 0 and 30-day old chicken, as determined by DNA-cellulose binding assay. However, nuclear binding of temperature and salt-activated GR complexes was significantly higher in 0-day old chicken. Cross-mixing experiments (wherein nuclei of day-0 were incubated with the H-R complexes of day-30 and vice-versa) revealed the role of nuclear specificity in higher binding of temperature and salt-activated H-R complexes at day-0 of postnatal age. DNase I extraction of nuclei bound to activated H-R complexes showed higher extractability at day-0 (70%), compared to day-30 (44%). Above findings suggested that changes in GR concentration as well as chromatin organization might play an important role in glucocorticoid-mediated responses during postnatal development of chicken.     

Kinetics of subcellular distribution in rat intestine of 1,25-dihydroxycholecalciferol administered in vivo. Evidence for concentration within 5 min into purified nuclei.

To better understand the initial steps in the induction of intestinal Ca2+ transport by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], we studied the early subcellular localization of 1,25(OH)2D3 in rat intestine. Vitamin D-deficient rats received 300 pmol of 1,25(OH)2[3H]D3 intravenously at 5 min to 4h before being killed. Cells homogenized in buffer of I = 90 mmol/litre were fractionated by centrifugation into a crude nuclear pellet, purified nuclei, Golgi and basal-lateral membranes, cytosol and a post-nuclear pellet. Nuclear purification was established by biochemical and morphological criteria and gave a yield of 32 +/- 2% (mean +/- S.E.M.; n = 21). Although re-establishment of Ca2+ uptake by Golgi is one of the earliest reported intestinal responses to 1,25(OH)2D3, no direct localization of 1,25(OH)2D3 to Golgi was detected. Purified nuclei had the highest specific radioactivity at all times studied, with nuclear localization detectable at 5 min and peak nuclear uptake at 1 h. Relative specific radioactivity of nuclei to cytosol increased from 5 min to 30 min, at which time equilibrium between cytosol and nucleus appeared to be attained. Nuclear uptake occurred in all cells from villus to crypt. Of total nuclear binding 10% was resistant to high ionic strength buffer (I = 365 mmol/litre); peak nuclear uptake was observed at 30 min in this buffer. This tight binding may represent the active fraction of 1,25(OH)2D3. These results indicate that localization of 1,25(OH)2D3 to rat intestinal nuclei precedes the observed Golgi-membrane effects and suggest the existence of high-affinity nuclear 1,25(OH)2D3-binding sites.     

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.     

Oestrogen receptors in chick oviduct. Characterization and subcellular distribution.

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

Sensitivity of progesterone receptors to aurintricarboxylic acid: Inhibition of nuclear uptake,ATP and DNA binding

When hen oviduct cytosol samples containing progesterone receptor complexed to [³H]progesterone were included with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose or ATP-Sepharose was abolished under simmilar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that auintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest the use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.     

Effect of colchicine on the depletion and replenishment of cytoplasmic glucocorticoid receptor in rat liver after administration of glucocorticoid

Pretreatment of rats with colchicine (3 mg/kg body weight) modified the time course of depletion of the cytoplasmic binding sites for 3H-dexamethasone after administration of prednisolone (0.5 or 1.5 mg/kg body weight). Colchicine also decreased the rate of the cytoplasmic receptor replenishment which was confirmed by application of this drug after completion of the cytoplasmic receptor translocation to nuclei (30 min after prednisolone injection). Addition of colchicine to the incubation mixture for in vitro binding of 3H-dexamethasone-labelled liver cytosol to isolated liver nuclei suspended in TKMS buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2 and 250 mM sucrose) evoked no measurable changes in the rate of the nuclear binding.